Abstract:
A process for producing a composition containing biologically active follistatin from a biological source, which composition is preserved and, especially pathogen free and is storage stable, preferably at room temperature. Further, the invention relates to the composition containing biologically active follistatin, which composition is available by the production process. The invention provides a process for producing a composition containing biologically active follistatin from avian eggs, or from raw animal blood serum.

Description:
PRIORITY CLAIM AND REFERENCE TO RELATED APPLICATION 
       [0001]    This application claims priority from EP 12171561.9, filed on 11 Jun. 2012 at the European Patent Office. 
       FIELD 
       [0002]    The invention concerns a method for producing a composition containing biologically active follistatin 
       BACKGROUND 
       [0003]    US 2007/0275036 A1 describes that follistatin is known to be present in fertilized avian eggs and is biologically active to increase muscle mass and to facilitate muscle regeneration in humans. Whereas the pasteurzation of liquid egg yolk is shown to inactivate the biological activity of follistatin contained therein, it is described that freeze-dried emulsified egg yolk can be irradiated e.g. by gamma radiation or by an electron beam for preservation without inactivating follistatin. 
       SUMMARY OF THE INVENTION 
       [0004]    The present invention relates to a process for producing a composition comprising biologically active follistatin from a biological source, which composition is preserved and, especially pathogen free and is storage stable, preferably at room temperature. Further, the invention relates to the composition containing biologically active follistatin, which composition is available by the production process. Follistatin has been found to he a secreted glycoprotein having activity to inhibit members of the TGF-β family, preferably to inhibit myostatin. Upon ingestion, the composition has activity to support, induce and/or positively regulate the increase of muscle in humans and animals. The composition is therefore suitable for use as a food ingredient or nutrition additive for humans and animals, e.g. for use as a compound for improving muscle increase and/or muscle regeneration. 
         [0005]    Preferably, the process for producing the composition, and the composition itself, are free from added chemical preservatives, most preferably, the process for producing the composition, and the composition, respectively, essentially consist of the natural components of the starting material, egg and its components, preferably egg yolk optionally including the white of egg, only subject to the physical treatment steps of the process. In the alternative to egg yolk, white of egg, which is also called egg white or egg albumen, and whole egg can be subjected to the steps of the process. In another preferred process, the biological source is raw animal blood serum. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0006]    Preferred embodiments of the invention are directed to a process for producing a composition containing biologically active follistatin from avian eggs or its components, especially from egg yolk, egg white or whole egg, the process comprising the preservation while maintaining a temperature below 38° C., preferably below 20° C., more preferably below 10° C., which step of preservation comprises or consists of subjecting the liquid egg yolk to a pressure of at least 4000 bar, for at least 1 minute, preferably to 5500-6500 bar, more preferably to 6000 bar for at least 1 minute, preferably for 3 minutes, more preferably for at least 5 minutes, preferably using an adiabatic compression and pressure release, and/or pulsed electric field treatment, preferably in a continuous process while pumping the liquid egg or its components, especially egg yolk, egg white or whole egg, through the space limited by at least 2 discharge electrodes, e.g. generating an electric field strength of 5 to 40 kV/cm, e.g. at 12 kV/cm at a flow rate of the liquid egg yolk of 30 L/h at a temperature of 30° C., preferably using unipolar pulses having a pulse duration of 5 to 20 μs, preferably of 10 μs, at a repetition rate of 70 to 200 Hz, especially positive, rectangular pulses. At an energy input of 50 to 140 kJ/kg, the decrease in bacterial contamination, determined as CFU, was by a factor of 10 to 630, respectively. 
         [0007]    The embodiments of the step of preservation are non-thermal process steps, i.e. an increase in temperature that may occur during the high pressure treatment and/or pulsed electric field treatment is not causative for the reduction in micro-organisms, especially of bacteria to achieve preservation. In addition, the embodiments of the step of preservation are physical treatment methods, i.e. without addition of antimicrobial chemical compounds. Accordingly, the embodiments of the step of preservation are non-thermal process steps consisting of physical treatment steps, which do not generate radicals and therefore maintain the chemical structure of the ingredients, especially of unsaturated fatty acids and vitamins of the composition. 
         [0008]    It was found that the high pressure treatment and/or the pulsed electric field treatment of liquid whole egg, liquid egg white, preferably of liquid egg yolk effectively reduces the bacterial contamination by a factor of at least 10, preferably by a factor of at least 100, more preferably of at least 1000. For example, for high pressure treatment, a reduction of the bacterial contamination to about 50 CFU/g, corresponding to a reduction by a factor of 3000 was found when starting from raw liquid egg yolk having a natural bacterial content of 1.5×10 5  CFU/g. For pulsed electric field treatment, a reduction by a factor of 10 to a factor of 1000 was found. The reduction of the natural microbiological contamination by the high pressure treatment and/or the pulsed electric field treatment is sufficient for preserving the egg white, whole egg or egg yolk. 
         [0009]    Preferably, the process subsequent to the preservation step comprises drying, especially freeze-drying of the liquid egg preparation, especially egg yolk, egg white or whole egg, resulting in an egg containing powder, especially an egg yolk, egg white or whole egg containing powder, preferably in a powder consisting essentially of the high pressure treated and/or pulsed electric field treated egg or egg constituents, e.g. egg yolk, egg white or whole egg. In the alternative to freeze-drying, the drying can be fluidized bed drying, preferably at a temperature at or below 42° C., preferably at or below 40° C., more preferably at or below 38° C. or at or below 35° C. 
         [0010]    It has been found that the process for producing the follistatin - containing composition comprising a preservation step comprising or consisting of high pressure treatment and/or pulsed electric field treatment, preferably with subsequent drying, especially but not limited to freeze-drying, leads both to an efficient reduction of bacterial contamination as determined e.g. as viable bacteria, and to the follistatin maintaining its biological activity, e.g. to at least 50%, preferably to at least 70%, more preferably to at least 80%, more preferably to at least 85%, at least 90% or to at least 95%, with reference to follistatin activity in the egg yolk composition used as the starting compound. 
         [0011]    Especially in view of preservation processes using irradiation, it is an advantage of the process of the invention that no radicals are generated by the step of preservation, and therefore the resulting preserved liquid egg yolk, egg white or whole egg, which preferably is subsequently dried, preferably freeze-dried, contains less or no radicals and reaction products of radicals. E.g. the preserved liquid egg yolk, egg white or whole egg, as well as the dried, preferably freeze-dried, preserved egg yolk, egg white or whole egg, contains unsaturated fatty acids of the egg yolk essentially in their natural state and composition, e.g. without changes to their double bonds. Accordingly, the composition obtainable by the process of the invention preferably contains the unsaturated fatty acids of egg yolk without changes of their double bonds, i.e. in their natural biological constitution. 
         [0012]    In the alternative to whole egg or egg yolk, the white of egg can be used in the process. 
         [0013]    Preferably, in the process, no chemical preservative is added, e.g. no anti-microbial agent is added. Optionally, an antioxidant is added, e.g. ascorbic acid or a neutral salt thereof. Preferably, the whole egg, egg white, more preferably egg yolk only is free from added ingredients, e.g. the whole egg, egg white, or more preferably the egg yolk, is subjected to the physical process steps only, which comprise, preferably consist of subjecting liquid whole egg, egg white or liquid egg yolk to high pressure treatment and/or to pulsed electric field treatment, preferably followed by drying, e.g. freeze-drying or fluidized bed drying. 
         [0014]    For high pressure treatment, it is preferred that the liquid whole egg, white of egg or liquid egg yolk is contained in sealed containers having an elastic wall, e.g. in plastic bags, more preferably free from gas, more preferably degassed. For a gas-free whole egg, egg white or liquid egg yolk in a container, gas bubbles can be expelled before sealing the container. For degassing, a reduced pressure can be applied prior to high pressure treatment, preferably also prior to pulsed electric field treatment. 
         [0015]    High pressure treatment is generally carried out using water as a compression medium that is pumped into a sealed chamber containing the liquid whole egg, egg white or liquid egg yolk until the high pressure is reached, maintaining the high pressure, and then releasing the pressure, e.g. by opening the high pressure container. 
         [0016]    It was found that after high pressure treatment within sealed containers, e.g. in sealed polyethylene bags, the liquid whole egg, egg white or liquid egg yolk preperation is stable, e.g. for 12 to 24 hours, preferably for 2 to 5 days, e.g. at 5 to 10° C., without a drastic increase in bacterial contamination, and especially without a significant loss of follistatin activity. 
         [0017]    For high pressure treatment, the adiabatic increase in temperature due to the high pressure preferably is counteracted by cooling the liquid whole egg, egg white or liquid egg yolk to a temperature which is at least 5° C., preferably about 10° C. below the maximum temperature, e.g. below 38° C. prior to the treatment. Preferably, prior to high pressure treatment and/or prior to the pulsed electric field treatment, the liquid whole egg, egg white or liquid egg yolk is cooled to a temperature of between 0 and 28° C., preferably to 5 to 20° C., more preferably to a maximum of 10° C. 
         [0018]    For pulsed electric field treatment, it was found that a short rise in temperature, e.g. to a maximum of 45° C., preferably to a maximum of 42° C. or to 40° C., for maximally 10s, preferably for maximally 5 or maximally 2s results in a low loss of active follistatin. Accordingly, for the pulsed electric field treatment, the aforementioned short rise in temperature is acceptable, although less preferred. 
         [0019]    Active follistatin was determined by size separation, e.g. by size-exclusion HPLC or by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), optionally followed by Western blotting and immunospecific detection using an anti-follistatin antibody. A reduction of the size-specific signal identified for follistatin in fresh yolk from fertilized eggs was used as an indicator for the reduction of follistatin activity, because an inactivation of follistatin results in the change, e.g. reduction of the molecule size. 
         [0020]    Preferably, the process comprises a step of concentrating the whole egg, egg white or egg yolk of the fertilized eggs. For concentrating, the fraction of whole egg, of egg white or of egg yolk having the higher proportion of follistatin is used, the fraction being obtained e.g. by size separation or by density separation. The preferred fraction is the fraction containing the egg yolk membrane, e.g. obtained from separating egg yolk or whole egg, and the fraction containing chalazae, e.g. obtained from separating the white of egg or whole egg. Preferably, the preferred fraction contains the major portion of the egg yolk membranes and/or of the chalazae of the whole egg, egg white or egg yolk subjected to the concentrating or separating step. For separating by size separation, sieving can be used, e.g. of a mesh size of 0.5 mm to 2 mm, preferably approx. 0.5 to 1 mm. Using size separation, the preferred fraction is the egg yolk membrane and/or chalazae containing fraction, which is the particulate or large fraction. For separating by density separation, centrifugation, e.g. using a centrifugal separator. Using density separation of whole egg, egg white or egg yolk, the higher density fraction is the preferred fraction. 
         [0021]    Optionally, prior to the step of concentrating the whole egg, egg white or egg yolk of the fertilized eggs by separating the fraction containing the egg yolk membrane and/or chalazae, the whole egg, egg white or egg yolk can be diluted to facilitate the separating step, e.g. using water as a diluent, the water optionally containing salt. 
         [0022]    In the alternative or in addition to whole egg, egg white or egg yolk of fertilized eggs, the process can be performed using blood serum from slaughtered animals as the starting material. Accordingly, the blood serum can replace the whole egg, egg white or egg yolk in the process, and therefore the description relating to whole egg, egg white or egg yolk also refers to blood serum. 
         [0023]    Optionally, the process can comprise the further step of mixing or encapsulating the dried preserved egg or egg constituent. Preferably, for mixing or encapsulating, the dried egg yolk, whole egg or egg white, or alternatively the dried blood serum, is admixed with a solution, preferably an aqueous solution of an encapsulating agent. The encapsulating agent can e.g. be a sugar, sugar alcohol and/or sugar polymer, a solution of which in the process is admixed with the preserved and dried egg yolk, whole egg or egg white, or alternatively the dried blood serum, and dried to produce encapsulated dried egg yolk, whole egg or egg white, or alternatively the dried blood serum. The sugar can e.g. be sucrose, fructose, glucose, and/or corn sirup. The sugar alcohol can e.g. be maltitol, isomalt etc. The sugar polymer can e.g. be starch, modified starch and/or cellulose and/or methylcellulose, which preferably also serves as an anti-caking agent. 
         [0024]    As a specific advantage of the high pressure treatment of liquid egg yolk, whole egg or egg white, it has been found that the bioavailability and digestability of the protein, preferably of the total protein, is enhanced. Therefore, the process comprising the step of high pressure treatment of liquid egg yolk, whole egg or egg white is preferred for producing a preserved composition containing biologically active follistatin, in which composition the protein has increased bioavailability, e.g. increased digestability, for example in relation to the non-treated liquid egg yolk, whole egg or egg white. 
         [0025]    Preferred embodiments of the invention are now illustrated non-limiting experimental examples. 
       EXAMPLE 1 
     Production of Preserved Egg Yolk Containing Active Follistatin 
       [0026]    Fertilized hen eggs ( gallas domesticus ) contained from a certified breeding station were used, which eggs were not brooded. The eggs were cracked and separated into egg yolk and the white of egg automatically. As raw liquid egg yolk, 3000 L egg yolk were used that were preferably homogenized by stirring were maintained at 5 to 10° C. and filled under sterile conditions into polyethylene bags and sealed after expulsion of entrapped air bubbles. These polyethylene bags could have a volume of between 1 L and 50 L, preferably of 5 to 20 L each. The bags were arranged in a high pressure chamber (NC-Hyperbaric, Spain). Using water as a pressurising medium, the pressure was increased to 6000 bar within 10 to 20 minutes. After a holding time of 3 or 5 minutes, respectively, the pressure was released by opening a release valve. 
         [0027]    The bacterial contamination was determined by standard dilution plating on complete medium and counting following cultivation in an incubator at 37° C. for 48 h. 
         [0028]    Aliquots from the high pressure treated egg yolk were kept at about 5° C. for a few hours and subsequently freeze-dried by freezing the egg yolk and applying vaccum to withdraw water, while controlling the temperature of the egg yolk to preferably not exceed 10° C., preferably 5° C., preferably keeping the egg yolk in a frozen state. 
         [0029]    The microbiological analysis showed that the high pressure treatment both for 3 minutes and 5 minutes resulted in a drastic reduction of bacterial contamination, and also the subsequent step of freeze-drying further reduced the bacterial contamination. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 bacterial contamination, measured as CFU/g 
               
             
          
           
               
                   
                   
                   Salmonella  in 
                 Total cell 
               
               
                   
                 sample 
                 25 g sample 
                 count (CFU/g) 
               
               
                   
                   
               
               
                   
                 raw liquid egg yolk 
                 Negative 
                 1.5 × 10 5   
               
               
                   
                 liquid egg yolk after 6000 
                 Negative 
                 50 
               
               
                   
                 bar, 3 min 
               
               
                   
                 liquid egg yolk after 6000 
                 Negative 
                 50 
               
               
                   
                 bar, 5 min 
               
               
                   
                 freeze-dried egg yolk after 
                 Negative 
                 40 
               
               
                   
                 6000 bar, 3 min 
               
               
                   
                 freeze-dried egg yolk after 
                 Negative 
                 &lt;10 
               
               
                   
                 6000 bar, 5 min 
               
               
                   
                   
               
               
                   
                 CFU = colony forming units (viable micro-organisms) 
               
             
          
         
       
     
         [0030]    Follistatin activity in the liquid egg yolk as determined by SDS-PAGE showed a reduction by approx. 15%, or a content of 85% active follistatin, on the basis of the content of active follistatin as determined by SDS-PAGE in the raw liquid yolk. 
         [0031]    In the freeze-dried egg yolk, the content of active follistatin in relation to the total protein concentration was the same as in the liquid egg yolk after high pressure treatment. This shows that the step of freeze-drying does not substantially affect the activity of follistatin, e.g. freeze-drying does not substantially reduce the concentration of active follistatin per total protein content. 
       EXAMPLE 2 
     Fraction of Freeze-Dried Egg Yolk Containing Active Follistatin Using Pulsed Electric Field Treatment 
       [0032]    An aliquot of the raw liquid egg yolk used in Example 1 was treated at a flow rate of 30 L/h at 30° C. by pulsed electric field of a field strength of 12 kV/cm using unipolar positive pulses having a pulse duration of 10 μs at a repetition rate of 200 Hz. At an energy input of 50 to 140 kJ/kg, the viable bacterial contamination was reduced by a factor of 10 and 630 CFU, respectively, as determined by dilution plating. 
         [0033]    Using SDS-PAGE, a reduction of active follistatin by approx. 15%, or a residual activity of follistatin of 85% based on the raw egg yolk was found. No thermal denaturation of the liquid egg yolk was observed in SDS-PAGE. 
       EXAMPLE 3 
     Concentrating Whole Egg, White of Egg or Egg Yolk by Separation 
       [0034]    The process of Example 1 was repeated with the alteration that before the high pressure treatment the egg yolk was separated by centrifugation at 3343×g for 20 min into a high density fraction that was collected as a pellet and a low density supernatant fraction. The high density was found the high follistatin fraction. 
         [0035]    In the alternative, whole egg or white of egg was separated by centrifugation at 3343×g for 20 min into a high density fraction that was collected as a pellet and a low density supernatant fraction. Again, the high density was found the high follistatin fraction. 
         [0000]    The analysis of the follistatin content is shown below: 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                 Follistatin 
               
               
                   
                 fraction 
                 [μg/kg] 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 white of egg, prior to centrifugation 
                 15 
               
               
                   
                 white of egg, pellet 
                 33 
               
               
                   
                 whole egg, prior to centrifugation 
                 23 
               
               
                   
                 whole egg, pellet 
                 41 
               
               
                   
                 egg yolk, prior to centrifugation 
                 4 
               
               
                   
                 egg yolk, pellet 
                 36 
               
               
                   
                   
               
             
          
         
       
     
         [0036]    These results show that the separation of egg yolk, whole egg or egg white to a higher density fraction, corresponding to egg yolk membranes and chalazae, results in an increased concentration of follistatin, which fraction after the step of preservation, preferably with subsequent drying, yields a composition having an increased follistatin concentration. 
         [0037]    Preferably, the egg yolk, whole egg or egg white prior to the separation was not homogenized, e.g. the egg yolk, whole egg or egg white was passed through a wide sieve or was stirred to only crack the egg yolk membrane to allow egg yolk to exit, preferably without breaking the egg yolk membrane or chalazae into small pieces. 
         [0038]    While specific embodiments of the present invention have been shown and described, it should be understood that other modifications, substitutions and alternatives are apparent to one of ordinary skill in the art. Such modifications, substitutions and alternatives can be made without departing from the spirit and scope of the invention, which should be determined from the appended claims. 
         [0039]    Various features of the invention are set forth in the appended claims.